Research conducted in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Intracellular protein degradation plays a critical part in controlling the levels of important cellular regulatory proteins and is an essential component of the protein quality control system. Most protein degradation within the cytosol is carried out by ATP-dependent proteases, which are multi-component molecular machines. The heart of the machine is an ATP-driven protein unfoldase that binds a specific protein target, disrupts its structure, and translocates the unfolded protein into the proteolytic chamber of a tightly associated self-compartmentalized endopeptidase. Our studies encompass structural and biochemical analysis of the ATP-dependent Clp and Lon proteases from E. coli and from human mitochondria and assay of their biological activities in cultured cells. In studies of human ClpXP, we found that hClpP and hClpX are required for cell viability. Human cells in culture die during the first 48 h after treatment with siRNA directed against either HCLPP or HCLPX. Intracellular hClpP protein levels decrease rapidly when synthesis is shut off, indicating that hClpP is unstable and must be continuous replenished to maintain ClpP activity in mitochondria. The decrease in hClpX protein is relatively modest but cell death occurs in nearly the same time as with HCLPP knockdown, suggesting that newly synthesized ClpX is needed for survival of cells. The decrease in enzymatic activity in cells treated with siRNA was shown with a fluorescent substrate whose levels respond to changes in hClpP or hClpX amounts. Green fluorescent protein (GFP) bearing a degradation tag (SsrA) for the bacterial ClpXP is degraded and undetectable in wild type cells. Knockdown of either hClpP or hClpX led to measurable GFP and more than 4-fold stabilization of the protein. Since, purified hClpXP cannot degrade GFP-SsrA, we propose that factors in mitochondria can stimulate the activity of human ClpXP or act as adaptor proteins to help deliver substrates to hClpXP. Short term treatment with HCLPP or HCLPX siRNA sensitizes the cells to apoptotic cell death in the presence of cisplatin or staurosporin. Over expression of human ClpP confers resistance to killing by the DNA-damaging agent, cisplatin, and by an inducer of the cell death receptor, TRAIL. Protection requires hClpP proteolytic activity and also the C-terminal domain of hClpP, a unique feature of mammalian ClpP proteins. We extended our studies of degradation of endogenous SsrA-tagged proteins in E. coli, which are produced when the tmRNA system detects stalled ribosomes and releases incomplete C-terminally tagged polypeptide chains. Using an anti-SsrA antibody prepared in our laboratory, we established that ClpXP plays the major role in degradation of SsrA-tagged proteins and that Lon and ClpAP have minor roles in degrading them. We showed that the tagging system operates at a high level when proteins are over expressed in cells and we were able to detect significant amounts of tagged over expressed proteins even in wild type cells. We are in the process of isolating endogenous SsrA-tagged proteins using the ability of mutant forms of ClpP to trap protein substrates in vivo. We have found by two-dimensional gels that there are fewer than 30-40 proteins that appear to be SsrA-tagged, suggesting that tagging does not occur equally for all translation reaction. We will isolate and identify SsrA-tagged substrates to determine what proteins are preferentially SsrA-tagged and whether tagging serves a regulatory function. We are conducting a study to define the parameters for the selective degradation of proteins bearing N-degrons in E. coli. We showed that proteins with non-canonical N-terminal amino acids (those not naturally exposed by activity of methionine amino peptidase) require ClpS, a small adaptor proteins that binds to the N-domain of ClpA, for degradation. We find that peptides with N-degrons are competitive inhibitors of protein degradation by ClpAP/ClpS and that adding methionine or serine to the N-terminus blocks the ability to inhibit ClpS activity. We are collaborating with Dr. Di Xia to crystallize N-degron peptide complexes with ClpS and ClpS bound to ClpA. We are mutagenizing conserved residues in the putative N-degron binding site of ClpS and will isolate and characterize the mutant proteins. To learn how proteins with N-degrons arise in cells, we have constructed mutants lacking other proteases that will retain the ability to specifically target N-degron proteins to ClpAP. We will express inactive ClpP to trap N-degron proteins, identify them by tandem mass spectrometry, and analyze for normal or abnormal N-terminal residues. Recoveries in the presence and absence of ClpS will be compared to determine if ClpS is needed for all N-degron targeting. Our structural studies are focused on novel aspects pertaining to substrate interaction and the response of the Clp complexes to substrate binding. ClpA undergoes a large conformational change upon binding of peptide substrates. In ultracentrifugation studies (done in collaboration with Dr. Grzegorz Piszczek, NHLBI) we have shown that binding of a decapeptide slightly reduces the sedimentation coefficient of ClpA and causes a dramatic narrowing of the distribution of structurally distinct species. Narrowed distribution indicates that peptide binding stabilizes a uniform conformation of ClpA and supports the hypothesis that peptides bind in the central channel of the hexamer. Peptide binding has the same effect on ClpA lacking the N-domain, indicating that the conformational flexibility is not due to the mobile N-domains. With ClpA lacking the C-terminal ATPase domain (D2), we found that the distribution was already narrow and not further narrowed by peptide binding. These results establish that peptides can migrate to ClpA-D2 and further suggest that the conformational variability of ClpA is due to motion in ClpA-D2. We purified mutants of ClpA with shorter linkers between the N-domain and ClpA-D1. ClpA with deletions of 10, 15, and 20 amino acids in the linker had activities similar to wild type. By electron microscopy (done in collaboration with Alasdair Seven, NIAMS) the ClpA N-domains were more visible when linkers were shorter, indicating that their range of motion was restricted. We will use ClpA hexamers with less mobile N-domain to map the position of ClpS bound to ClpA and to visualize substrates bound to the ClpS/N-domain complex. The N-terminal peptides of ClpP affect activity and interact with ClpA and ClpX. We expressed ClpP lacking a portion of the N-terminal loop and thus has a larger axial channel. This form of ClpP has greatly enhanced peptidase activity in the absence of activation by ClpX or ClpA. Activity is nearly 40% of fully activated wild-type ClpP. We do not know whether increased activity is due to the increased size of the channel , allowing peptides into the chamber of ClpP more rapidly, or to increased activity at the proteolytic site due to allosteric communication between the N-terminus and the active site. Surprisingly, ClpX and ClpA inhibit the activity of ClpP when the N-terminal loop is deleted. Inhibition s [summary truncated at 7800 characters]